Within cells proteins are targeted to well-defined locations by specific interactions with cellular constituents such as proteins and phospholipids. During the late phase of HIV-1 infection, Gag polyproteins are transported to the plasma membrane (PM) for assembly. Using nuclear magnetic resonance (NMR) we have previously defined the molecular basis of one component of Gag targeting to the PM, the interaction of the matrix (MA) domain with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Our preliminary data indicate that Gag also forms biologically important interactions with cellular lipids and proteins implicated in efficient particle assembly. We show that MA interacts directly with phosphatidylserine (PtdSer), phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn), suggesting that Gag targeting and assembly on the PM proceed via a dual engagement of the MA domain with PI(4,5)P2 and major membrane lipids. A number of cellular proteins have been proposed to facilitate Gag intracellular trafficking and targeting to the PM. HIV-1 Gag was shown to interact and co-localize with calmodulin (CaM) in the cytoplasm. In addition, more recent studies have identified the suppressor of cytokine signaling 1 (SOCS1) as an inducible host factor during HIV infection, which regulates the late stages of the virus replication pathway through direct interactions with the Gag protein. However, the molecular mechanism by which Gag interacts with these factors has yet to be established. Preliminary studies demonstrate that HIV-1 MA interacts directly with CaM in a calcium- dependent manner, suggesting that HIV-1 hijacks CaM cell-signaling pathway to facilitate Gag trafficking. In addition, we have obtained evidence for direct interactions between SOCS1 and the MA protein. In this proposal, we will employ biochemical, biophysical and structural biology tools to identify key protein-protein and protein-lipid interactions involved in the molecular mechanism governing HIV-1 Gag intracellular trafficking and assembly. Our three main aims are: (i) to determine the precise molecular mechanism for Gag binding to the PM. We will determine at the structural level how various lipid constituents interact with the MA protein and will identify the functional importance of the hydrophobic insertion, specific and non- specific electrostatic interactions, (ii) to identify the functional role of CaM in Gag trafficking and assembly, and (iii) to elucidate the structural requirements for SOCS1-Gag interactions. The results generated by this research will offer a better understanding of how HIV-1 Gag interacts with cellular constituents at the atomic level, which could lead to new approaches to rational design of new antiviral therapeutic agents that inhibit Gag trafficking and assembly. PUBLIC HEALTH RELEVANCE: HIV remains the most deadly pathogen that threatens human's life and the lack of efficient cure poses a major problem in public health and safety. Major efforts are now being focused on understanding how HIV interacts with the host cell during the replication cycle. The proposed studies will help in understanding the precise molecular mechanism of HIV Gag trafficking and assembly, which may aid in the development of new strategies for antiviral drug design.